Method for affinity purification

ABSTRACT

The disclosure relates to a switchable aptamer having a high affinity for a selected target such as a virus, cell or antibody when in the presence of a binding ion and a low affinity for said target in the absence of said binding ion. The switchable aptamer may be isolated or selected from a pool comprising a mixture of aptamers by incubating the pool with the target ligand and a binding ion to form target-aptamer complexes; separating unbound aptamer molecules from the target-aptamer complexes; contacting the target-aptamer complexes with a chelating agent having affinity for the binding ion wherein a switchable aptamer specific to said target is released from the target-aptamer complexes; and isolating the switchable aptamer released in the preceding step.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 14/277,110,filed May 14, 2014, which in turn claims the benefit of application No.61/823,638 filed on May 15, 2013. The contents of said priorapplications are incorporated by reference into the present application.

FIELD

The present disclosure is in the field of chemical processes, namelyaffinity purification of target ligands capable of binding to switchableaptamers. The disclosure further relates to the isolation of switchableaptamers for targeting cells, viruses and antibodies.

BACKGROUND

The Systematic Evolution of Ligands by EXponential enrichment method, orSELEX, is a combinatorial chemistry technique for producingoligonucleotides of either single-stranded DNA or RNA that specificallybind to one or more target ligands. The method involves selection from amixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve a desired level of binding affinity andselectivity. SELEX has been used to evolve nucleic acid aptamers ofextremely high binding affinity to a variety of targets. Some of thesetargets include, for example, lysozyme (Potty et al.), thrombin (Long etal.), human immunodeficiency virus trans-acting responsive element (HIVTAR) (Darfeuille et al.), hemin (Liu et al.), interferon gamma (Min etal.), vascular endothelial growth factor (VEGF) (Ng et al.), prostatespecific antigen (PSA) (Savory et al.; Jeong et al.).

Aptamers have found applications in many areas, such as biotechnology,medicine, pharmacology, microbiology, and analytical chemistry,including chromatographic separation and biosensors.

Interestingly, structure-switching aptamers or SwAps have also foundmultiple applications. A review of SwAps as biosensors was published in2009 by Sefah et al. Changes in fluorescence intensities between freeand bound aptmamer complexes have been described to detect cocaine byaptamer-based capillary zone electrophoresis (Deng et al.). Multiplesmall molecule analytes have been detected by a similar method (Zhu etal.) High surface area, solid phase sol-gel-derived macroporous silicafilms have also been shown to be suitable platforms for high-densityaffinity-based immobilization of functional single stranded-aptamermolecules, allowing for binding of both large and small target ligandsthrough SwAps with robust signal development (Carrasquilla et al.)

Aptamers have further been used for protein and small moleculepurification using affinity chromatography. Indeed, aptamer affinitychromatography has been applied to protein purification (Romig et al.)and in the separation of mature dendritic cells from immature dendriticcells (Berezovski et al.). However, aptamer affinity chromatography hasnot to the inventors' knowledge been shown in the prior art to apply tothe purification of cells, viruses or antibodies.

A major problem encountered when dealing with aptamer-based affinitychromatography to purify target ligands such as viruses, cells andcertain other biological materials is the need for elevated temperaturesor the addition of detergents to alter the conformation of the SwAp andto subsequently allow the release of the captured biomolecular targetligand from the solid medium or chromatography column. These harshregeneration techniques decrease significantly the viability of cellsand viruses, denature proteins and irreversibly change the structure ofbiomolecules. Furthermore, the lack of an efficient regenerationtechnique that can be generalized to other target-specific aptamers hasbeen a challenge to the widespread use of aptamers for purification.Concerns have also been raised with regard to the possiblecross-reaction between aptamers and other contaminants that might existin the mixture containing the biomolecule to be purified. As such, untilnow, these problems have made the utilization of aptamers for thepurification and recovery of purified targets such as viruses and cellsvery difficult to achieve.

The methods currently available for purification of viruses include:differential centrifugation, size exclusion chromatography (SEC) andheparin affinity column chromatography. These techniques are not withoutchallenges. Sucrose differential gradient centrifugation is conventionalfor virus isolation in small quantities, but it is difficult toscale-up, is labour-intensive and requires long processing times, whichmay decrease the infectivity of viruses (Diallo et al.) SEC does notseparate well from cell debris or large molecular aggregates withsimilar sized viruses, and is followed with additional concentrationsteps such as ultrafiltration or polyethylene glycol-6000 precipitation.The heparin column purification utilizes sepharose beads conjugated tolinear anionic heparin molecules. This technique is used to purifyproteins containing a heparin-binding domain as well as retroviruses.Although, this heparin method yields a purer product than the densitygradient method, it still requires additional SEC purification fromcationic proteins and salt.

SUMMARY

The present disclosure is directed to the purification of a targetligand of interest using aptamer molecules which exhibit a switchableaffinity for the target in the presence or absence of a binding ion.According to various embodiments, the target can be a virus, aeukaryotic cell which may be receptor-positive for a selected receptor,a prokaryotic cell or an antibody.

According to one aspect, the disclosure relates to a method of isolatinga switchable aptamer having affinity for a selected target ligand from apool comprising a mixture of aptamers. The mixture may consist of arandomized pool of aptamers. According to this aspect, the methodcomprises the steps of:

a) incubating said pool with said target and a binding ion to formtarget-aptamer complexes comprising said target and aptamers specific tosaid target;

b) separating unbound aptamer molecules from the target-aptamercomplexes;

c) contacting the target-aptamer complexes with a chelating agent havingaffinity for said binding ion wherein a switchable aptamer specific tosaid target is released from the target-aptamer complex; and

d) isolating the switchable aptamer released in step c.

The present method may comprise selecting the switchable aptamer.

At least steps a through c may be performed at room temperature, forexample a maximum temperature of 25° C.

The method may comprise the further step of amplifying the switchableaptamer isolated in step d.

The method may comprise the further step of measuring the affinity ofsaid switchable aptamer for the target in the presence and absence ofthe binding ion.

Another aspect relates to an iterative process wherein two or moreswitchable aptamers are isolated and steps a through d are repeatedusing the two or more switchable aptamers in place of the mixture ofaptamers in said pool wherein a switchable aptamer is isolated which hasan increased affinity for the target relative to others of saidswitchable aptamers. From about 5 to about 20 such iterations or rounds(or between 5 and 20) may be performed for sequentially achieving higherpurification levels, or from about 7 to about 15 (or between 7 and 15)rounds, or about 10 rounds.

Suitable targets for the method include a virus such as VesicularStomatis Virus (VSV) or a cell. Cellular targets include a receptorpositive cell for a selected receptor such as a Neuropilin 1 (NRP)receptor, a Leukemia inhibitory factor (LIF) receptor, a Patched 1(PTCH1) receptor, a Delta-Like Ligand 4 (DLL4) receptor or a plasminogenactivator/urokinase receptor (PLAUR). A prokaryotic cell such as abacteria may also comprise a target.

The binding ion may be a monovalent or divalent ion. Suitable divalentions include calcium or magnesium or a combination thereof.

Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA)or ethylene glycol tetraacetic acid (EGTA) or a combination thereof.

The target-aptamer complexes and the unbound aptamer molecules may beseparated by centrifugation or by immobilizing the target-aptamercomplexes and washing away unbound aptamer.

Suitable aptamers comprise nucleotides. The aptamers in said pool maycomprise a random region of between 20 and 60 (or from about 20 to about60) nucleotides, for example about 40 nucleotides.

According to a further aspect, the disclosure relates to a switchableaptamer having a high affinity for a selected target in the presence ofa binding ion and a low affinity for said target in the absence of saidbinding ion. The switchable aptamer may be obtained through the methoddescribed herein. The switchable aptamer may comprise the nucleotidesequence represented in any one of SEQ ID NOS: 3 through 17 or 21through 89, or a nucleotide sequence with at least 70%, 75%, 80%, 85%,90%, 95% or 99% identity with any one of SEQ ID NOS: 3 through 17 or 21through 89. The aptamer may have a target as described above.

According to a further aspect, the disclosure relates to a method forpurifying a selected target from a complex mixture. According to thisaspect, the method comprises the steps of:

a) providing a switchable aptamer having affinity for said targetwherein the switchable aptamer has a high affinity for the target when abinding ion is present and a low affinity for the target when thebinding ion is absent;

b) incubating said switchable aptamer with the complex mixture in thepresence of said binding ion to produce target-aptamer complexes;

c) separating the target-aptamer complexes from unbound components ofthe complex mixture;

d) adding a chelating agent having affinity for the binding ion to thetarget-aptamer complexes to release the target from the target-aptamercomplexes; and

e) separating the released target from said switchable aptamer.

Suitable conditions and targets for said method may be the same asdescribed herein for the method of isolating the switchable aptamer. Themethod may be performed at room temperature (maximum of 25° C.).

The method may comprise the further step of re-using the separatedswitchable aptamer to repeat said steps a through e on the same or adifferent complex mixture.

The switchable aptamer used in said method may comprise the switchableaptamer isolated according to the method described herein.

In the present specification, the following definitions apply, unlessthe context clearly requires otherwise:

“SwAps” or “switchable aptamers” means an aptamer with switchableaffinity for one or more target ligands, wherein the affinity may beselectively increased or decreased by changing the environment of theaptamer such as contacting the aptamer with one or more ions.

“Target ligand” or “target” means a virus, a prokaryotic cell, aeukaryotic cell an antibody or a biomolecule whether synthetic ornaturally produced which is capable of being bound to an aptamer.

“Biomolecule” means any molecule that is produced by a living organismor which is a synthetic analogue of such a molecule, including largemacromolecules such as proteins, protein antibodies, peptidespolysaccharides, lipids, and nucleic acids, as well as small moleculessuch as primary metabolites, secondary metabolites, and naturalproducts.

“Aptamer” means a molecule that has an affinity for and binds to aspecific target molecule via an interaction between the nucleic acidsequence of the aptamer and the target. This base pairing createssecondary structures such as short helical arms and single strandedloops. Combinations of these secondary structures results in theformation of tertiary structures that allow aptamers to bind to targetsvia van der Waals forces, hydrogen bonding and electrostaticinteraction—aligning with the same ways antibodies bind to antigens.

When this tertiary structure forms, the entire aptamer folds into astable complex with the target ligand.

An aptamer may constitute a nucleic acid, which may be RNA or DNA or apeptide for a peptide aptamer.

“Chelating agent” is a molecule which binds to a metal ion. Chelationinvolves the formation or presence of two or more separate coordinatebonds between a polydentate (multiple bonded) ligand and a singlecentral atom. Usually these ligands are organic compounds, and arecalled chelants, chelators, chelating agents, or sequestering agents.

“Binding ion”—an ion participating in formation aptamer-target complex

“Complex mixture”—a mixture of a target and non-target molecules,viruses, cells, and particles.

The objects and advantages of the present disclosure will become moreapparent upon reading the following non-restrictive description of thepreferred embodiments thereof, given for the purpose of exemplificationonly, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a flow chart of the selection protocol for switchableaptamers through modified cell-SELEX.

FIG. 1B is a schematic representation of virus purification byswitchable affinity aptamers.

FIG. 2A shows a bar graph of the binding of the to pools and controlaptamers to VSV in the presence of Ca²⁺ and Mg²⁺ (bars marked “+”) andthe amount of VSV remained bound to each pool after incubation withEDTA/EGTA mixture (bars marked “0”).

FIG. 2B shows a bar graph of Coefficient of Switching (CoS) valuescalculated for each of the ten (10) aptamer pools by flow cytometry.

FIG. 2C shows flow cytometry histograms of aptamer pool 10 and Round 0.The left panel shows strong binding aptamer pool used to begin theexperiment and the right panel shows aptamer pool 10 exhibitingswitchable behaviour.

FIG. 3A shows a bar graph of the binding affinities of aptamer clones(50 nM) incubated with VSV (107 PFU) for 30 min prior to separation into2 fractions one in DPBS (MgCl₂ and CaCl₂) (bars marked “+”) and onecontaining 2.5 mM EDTA/EGTA (without MgCl₂ and CaCl₂) (bars marked “0”).

FIG. 3B is a bar graph showing the Coefficient of Switching (CoS) of 15aptamer clones obtained using flow cytometry.

FIG. 4A is a schematic diagram of the electrochemical sensor developedto measure the affinities of the developed SwAps to VSV and thecoefficient of switching.

FIG. 4B is a plot of the change of resistance to charge transfer (RCT)after VSV binding (affinity indicator) and coefficient of switching(CoS) for each switchable aptamer.

FIG. 5A shows cyclic voltammograms recorded at a scan rate of 100 mVs⁻¹, where (a) bare GNPs-SPCE; (b) after coating with the hybridizedproduct of thiolated primer and VSV-specific switchable aptamer; (c)after surface back-filling with 1 mM 2-mercaptoethanol. FIG. 5B showsNyquist plots (−Zim vs. Zre) of impedance spectra of the VSV aptasensorafter each immobilization or binding step in 25 mM sodium phosphatebuffer (pH 7), containing 2.5 mM K₄Fe(CN)₆ and 2.5 mM K₃Fe(CN)₆.

FIGS. 6A-6P show Nyquist plots (−Zim vs. Zre) of impedance spectra ofVSV aptasensors based on 15 aptamer clones (Swaps1→Swaps15) obtained (a)after aptasensor preparation (b) after binding of 1×10⁶ PFU of VSV inDulbecco's phosphate buffered saline (DPBS), and (c) after treatmentwith an equimolar mixture of EDTA and EGTA (50 mM).

FIG. 7 shows a bar chart of the binding affinity of SwAp clone 6 indifferent buffers.

FIG. 8A is a scatterplot containing both VSV and Vero cell debrisobtained from a plate used for virus harvesting.

FIG. 8B shows a scatterplot of the purified VSV product obtained usingSwAps.

FIG. 8C is a graph of the fluorescence corresponding to VSV as shown inFIG. 8B (first peak), and VSV bound to Alexa-488-labeled anti-VSVantibodies (second peak).

FIG. 9 shows the results of the plaque-forming assay of infection by VSVafter SwAps-based purification. Panel 1 shows DPBS alone. Panel 2 showsinfection of 100 PFU from stock VSV and Panel 3 shows VSV retrieved uponcompletion of the purification.

FIGS. 10A-F show flow cytometry data relating to cloning and sequencingof high affinity SwAps to Neuropilin 1 (NRP) receptor.

FIGS. 11A-J show flow cytometry data relating to cloning and sequencingof high affinity SwAps to Leukemia inhibitory factor (LIF) receptor.

FIGS. 12A-G show flow cytometry data relating to cloning and sequencingof high affinity SwAps to Patched 1 (PTCH1) receptor.

FIGS. 13A-F show flow cytometry data relating to cloning and sequencingof high affinity SwAps to Delta-Like Ligand 4 (DLL4) receptor.

FIGS. 14A-I show flow cytometry data relating to cloning and sequencingof high affinity SwAps to plasminogen activator, urokinase receptor(PLAUR).

FIGS. 15A-I show PLAUR titration data.

While the disclosure will be described in conjunction with exampleembodiments, it will be understood that it is not intended to limit thescope of the disclosure to such embodiments. On the contrary, it isintended to cover all alternatives, modifications and equivalents as maybe included and defined by the appended claims.

DETAILED DESCRIPTION

The disclosure provides for the selection and purification of SwAps withselectively variable binding to a target and the use of such SwAps forthe purification of the target from a complex mixture. In general, thiscan be achieved by the steps of:

-   a) isolating an aptamer with switchable affinity to the selected    target from a nucleic acid or peptide library;-   b) contacting the target and the isolated aptamer in the presence of    a binding ion to produce a mixture of target-aptamer complexes;-   c) separating the target-aptamer complexes from the complex mixture;-   d) adding a binding ion chelator to the target-aptamer complexes to    separate the binding ion from the target/aptamer complex and thereby    trigger affinity switching; and-   e) collecting the released target, and optionally, independently    collecting the aptamer with switchable affinity to the target and    the purified target.

In one aspect, the present method for the purification of the targetusing SwAps assumes that the target needs to be purified from a complexsolution. The complex solution may comprise a mixture of biologicalmolecules and may also contain non-biological molecules. For example,the target may be in a solution containing debris and impurities.

In one aspect, the target is a virus, such as VSV. It will be understoodthat the virus can also be a virus other than VSV.

Step a) involves providing a library of randomized nucleic acidsequences and isolating an aptamer with a relatively high degree ofswitchable affinity to a specific target from this pool, in which thepool has variable degrees of affinity for the target. In someembodiments, the randomized library comprises a mixture of differentnucleic acids, each of which has a region of about 20-60, preferablyabout 40 randomized nucleotides as set out in SEQ ID NO: 18. Otherlengths of the randomized region are from 15 to 100 nucleotides. Thedesired aptamers to be isolated are those with switchable affinity tothe target in the presence or absence of a binding ion. This step a) maybe performed separately from the purification, or as the first step inpurifying the target of interest.

Isolation of the switchable aptamer is performed through a modifiedcell-SELEX process. More specifically, a pool of randomized aptamers isincubated with the target for a length of time, such as 30 minutes, inthe presence of binding ions (such as Ca²⁺, Mg²⁺, or a combination ofCa²⁺ and Mg²⁺) to produce an aptamer-target mixture. This mixture iswashed to remove any unbound aptamers. In some embodiments, the unboundaptamers are removed by centrifugation, leaving only the aptamers, whichare bound to the target in the mixture. In other embodiments, theunbound aptamers are washed away from immobilized aptamer-targetcomplexes using a washing agent.

The mixture is then subjected to treatment with a chelating agent, forexample ethylenediaminetetraacetic acid (EDTA) and/or ethylene glycoltetraacetic acid (EGTA). The chelating agent will be chosen inaccordance with the binding ion. For example, EDTA may be used tochelate Ca²⁺ ions, whereas EGTA may be used to chelate Mg²⁺ ions. Theaddition of the chelating agent and the corresponding reduction in freebinding ions induces a conformational change in some aptamers in therandomized pool, which allows them to become unbound from the target.Such aptamers with variable target affinity to the target are examplesSwAps.

The unbound SwAps are then separated from the target and target-aptamercomplexes. In some embodiments, the chelated mixture is centrifuged toremove the unbound target and the non-switchable aptamers still bound tothe target. In other embodiments, the unbound SwAps are separated bywashing or elution from target and target-aptamer complexes immobilizedon a surface. The SwAps are then amplified, for example by polymerasechain reaction (PCR), thereby enriching the SwAps within the aptamerpool. These amplified SwAps can then be re-isolated any number of timesto further enrich the aptamer pool for SwAps with switchable affinity tothe target.

In some embodiments, the steps of incubating the pool, separating thecomplexes, chelating the complexes, and separating the released SwAps isconducted at room temperature. This is especially preferred where thetarget is temperature sensitive, such as where the target is a virus ora cell. Amplification can be conducted at higher temperatures, asdictated by the polymerase chain reaction (PCR) protocol used.

Optionally, the selection method may also include an assay of thebinding affinity of the SwAp to the target in the presence or absence ofthe binding ion. Such assessments may be useful for the identificationof SwAps of particular interest for purification of the target,particularly those which exhibit a large change in affinity in thepresence or absence of the binding ion. Affinity measurements alsopermit the selection to be conducted in an iterative fashion until aSwAp having a desired affinity is obtained. In one embodiment, theaffinity assay is performed using flow cytometry. In other embodiments,target affinity may be measured using electrochemical means, such as byimpedimetric assays. Each of these affinity assays are described infurther detail below. Affinity assays may also be carried out in avariety of other ways known to the person of skill in the art. Examplesinclude use of a gel-shift assay, filter-binding assay, surface plasmonresonance, stopped-flow assay or isothermal calorimetry.

For example, as discussed further below, an aptamer isolated for thepurification of VSV may comprise any one of the nucleotide sequences setout in SEQ ID NOs: 3 to 17, each of which were derived from theselection method according to the present disclosure. In anotherembodiment, the aptamer has a sequence which has at least 70%, 75%, 80%,85%, 90%, 95% or 99% identity with any one of SEQ ID NOs: 3 through 17.

In one embodiment, SwAps that have been isolated in Step a) are labelledwith a tag. This tag is, for example, biotin. Other tags such asfluorescent dyes and markers are also contemplated.

In further embodiments of the disclosure, SwAps isolated in Step a) areimmobilized on a surface, such as the stationary phase of achromatography column, on a magnetic bead, on a membrane or in an agarmedium prior to being contacted with the target. For example, asdiscussed further below, one or more biotin labelled aptamers may beimmobilized on streptavidin-coated magnetic beads. Various other meansof immobilizing SwAps would be apparent to those of skill in the art.For example, SwAps may be immobilized on a glass substrate modified withorganosilanes or other fixing agents. Gold surfaces may also be used inconjunction with thiol-modifications to immobilize SwAps. A variety ofother physical adsorption, covalent bonding, affinity binding, andmatrix entrapment techniques are known in the art.

Immobilization of the SwAps aids in the separation and washing stepsduring purification, particularly in respect of the separation of theaptamer-target complexes from the complex solution and the recovery andre-use of SwAps for further rounds of purification. For example, a SwApimmobilized on a magnetic bead may form aptamer-target complexes whichcan be separated from the complex solution as shown schematically inFIG. 1B. Similarly, as shown in FIG. 1B, magnetic beads may also be usedto separate chelated SwAps from the target molecule. In otherembodiments, immobilization of SwAps on the stationary phase of achromatography column, in an agar medium, or on a membrane may be usefulfor separating aptamer-target complexes from the complex solution orchelated SwAps from the target by elution.

In Step b), the SwAps obtained through the isolation in Step a) areincubated with a binding ion. The binding ion can be, for example, adivalent ion like calcium or magnesium. Both calcium and magnesiumtogether can also act as the binding ion. It will be understood that amonovalent or divalent cation can be used as the binding ion.Submillimolar levels of the binding ion (0.01-1 mM) inducesconformational changes in the aptamer DNA and stabilize secondary andtertiary structures of the switchable aptamers. In the early foldingstages, aptamers form secondary structures stabilized through thebinding of monovalent cations or divalent cations in order to neutralizethe polyanionic backbone. The later stages of this process involve theformation of DNA tertiary structure, which is stabilized almost largelythrough the binding of divalent ions such as magnesium and calcium withcontributions from potassium binding. As such, the SwAps bind to theirtargets in the presence of Mg²⁺ and Ca²⁺ ions and release their targetsonce the ions are removed by the addition of the binding ion chelator inStep d).

Step c) calls for the separation of the target-aptamer complexes of Stepb) from the complex mixture. Where the SwAps are immobilized, thistypically involves washing the target-aptamer complexes with a washingagent to remove debris or impurities. For example, the washing agent canbe Dulbecco's phosphate-buffered saline (DPBS), which is particularlyuseful where the binding ion is Mg²⁺ or Ca²⁺. If the aptamers are notimmobilized, other means of separation known in the art may be employed,such as centrifugation or electrophoresis.

The above steps result in a highly purified aptamer/target complex, suchthat the subsequent separation of these components produces essentiallya two-component mixture comprising an isolated target and an isolatedswitchable aptamer that is specific to this target.

Step d) describes the addition of a chelating agent to the mixture oftarget-aptamer complexes to chelate the binding ion. The chelating agentwill be selected in accordance with the binding ion. For example, if thebinding ion is Ca²⁺, the binding ion chelator will beethylenediaminetetraacetic acid (EDTA). Similarly, if the binding ion isMg²⁺, the binding ion chelator will be ethylene glycol tetraacetic acid(EGTA). It follows that if Ca²⁺ and Mg²⁺ are both used together asbinding ions, both EDTA and EGTA will be used a chelators. The chelatorsremove the binding ion, and consequently allow for the release of thetarget by the SwAps.

Step e) describes the collection of the purified target released bychelation of the SwAps. In some embodiments, such as the embodiment showin FIG. 1B, this is accomplished by precipitating the SwAps out ofsolution using magnetic beads. In other embodiments, SwAps coupled toparticulate surfaces may be drawn out of solution by centrifugation. Instill other embodiments, SwAps are retained in the stationary phase of achromatography column while the target is eluted using a washing agent.In still other embodiments, SwAps are retained on the surface of asubstrate (such as, for example, agar, glass, or gold) and the releasedtarget is collected from solution. Optionally, the aptamer withswitchable affinity is also collected or retained, thereby permittingthe aptamer to be re-used a number of times for the purification of thesame target.

In some embodiments, the purification process is performed in aniterative manner to achieve higher levels of purity, such that thepurified target from the first round of purification acts as the complexsolution in subsequent rounds of purification.

EXAMPLES

The following examples detail the use of SwAps with controlled affinityfor the purification of Vesicular Stomatis Virus (VSV). As shown below,the virus captured with such SwAps can be recovered upon treatment witha simple eluent, containing a mixture of EDTA/EGTA at room temperatureand neutral pH. Using the method described herein, a total of 15sequences were obtained as a first pool of aptamers with varyingaffinity to VSV. Each sequence was assessed for affinity andswitchability by both flow cytometry and impedimetric analysis. SwApsclones 5, 6, 7 and 9 were selected for further study. SwAps clone 6 wasthe candidate that demonstrated the best affinity and switchability forVSV. The aptamers switchability is a function of the divalent cationsand the affinity of the SwAps to VSV can be terminated upon chelation ofthese cations. The resulting system provides an efficient and efficiencyand simplicity of the SwAps-based purification technique described hereabove.

Example 1: Production and Purification of VSV

The protocol of VSV production and harvesting was previously describedelsewhere (Diallo et al.). Briefly, 6 plates of Vero cells were grownuntil confluent and then were infected with VSV (approx. 10⁶ PFU/plate).After 24 hours, the supernatant was collected into 50-mL tubes andcentrifuged to removed the cell debris. Subsequently, the supernatantwas passed through a 0.2 μm filter (Pall Inc., USA) and the pelletcontaining the virus was then re-suspended in DPBS. Finally, it wasaliquoted and stored at −80° C.

Example 2: DNA Library and Primers

N40 ssDNA library, with the sequence(5′CTCCTCTGACTGTAACCACG-(N₄₀)-GCATAGGTAGTCCAGAAGCC3′) (SEQ ID NO:90) wasused for all experiments. It consists of total of 80 nucleotides andcontains two flanking primer regions of 20 nucleotides each, whereas thecentral region contains 40 random nucleotides. Fluorescently-labeled5′-primer (6-FAM/5′CTCCTCTGACTGTAACCACG3′) (SEQ ID NO:1) and the nonlabeled 3′-primer (5′GGCTTCTGGACTACCTATGC3′) (SEQ ID NO:20) and thelibrary were all obtained from Integrated DNA Technologies, U.S.

After the selection process described herein, some aptamers can beslightly longer or shorter due to mutations happened in PCR steps ororiginal synthesis of the DNA library.

The larger the size of this region, the more combinations of nucleotideswill result and the greater the chance of an exact “hit” with a target.In some embodiments, practical limits for are about 20-60 nucleotidesfor the central region.

Example 3: Selection of Aptamers with Switchable Affinity

Submillimolar levels of calcium and magnesium (0.01-1 mM) induceconformational changes of DNA and stabilize secondary and tertiarystructures. In the early folding stages, aptamers form secondarystructures stabilized through the binding of monovalent cations ordivalent cations in order to neutralize the polyanionic backbone. Thelater stages of this process involve the formation of DNA tertiarystructure, which is stabilized almost largely through the binding ofdivalent ions such as magnesium and calcium with contributions frompotassium binding. Here the use of these cations through a modifiedcell-SELEX technique to create aptamers with switchable affinity hasbeen exploited. These aptamers have a switchable functionality allowingthem to bind to their targets in the presence of Mg²⁺ and Ca²⁺ ions andto release their targets once the ions are removed. To allow for thisfunctionality, an additional step in the cell-SELEX process was addedusing two strong chelating agents, namely 2.5 mM EDTA and EGTA in PBS.The SwAps selection scheme through modified cell-SELEX is presented inFIG. 1A.

The process for the selection of SwAps involves 5 steps: 1) Incubationof aptamers with 2.5×10⁹ pfu mL⁻¹ of VSV in DPBS for 30 minutes; 2)Washing VSV to remove unbound aptamers by centrifugation; 3) Treatmentwith EGTA & EDTA to remove Mg²⁺ and Ca²⁺ and release VSV; 4) Collectionof unbound switchable aptamers through centrifugation; and 5) PCRamplification (symmetric+asymmetric).

In more ample details, the selection of the switchable aptamers beginswith adding VSV to a pool of aptamers in DPBS, along with Ca²⁺ and Mg²⁺,followed by incubation for 30 min at room temperature. This allowed forbinding between aptamers and VSV to reach equilibrium.

Centrifugation allowed for the removal of unbound DNA aptamers, wherethe aptamers bound to VSV became a part of the pellet after thecentrifugation step, whereas the non-bound DNA remained in thesupernatant and was discarded. Addition of EGTA & EDTA to remove Ca²⁺and Mg²⁺ allowed for the collection of aptamers exhibiting switchableaffinity. These aptamers were collected and amplified by symmetric andasymmetric PCR.

The selection scheme involved 5 steps and was repeated for 10 rounds.The scheme involved (1) incubation of aptamer pool with VSV, (2)separation of bound aptamers, (3) addition of EDTA and EGTA, (4)collection of unbound aptamers, and (5) PCR to amplify the desiredaptamer pool. Each aptamer pool was denatured by heating at 95° C. for 5min in Dulbecco's phosphate buffered saline (DPBS), containing 0.901 mMCaCl₂, 0.493 mM MgCl₂, 2.67 mM KCl, 137.93 mM NaCl, 1.47 mM KH₂PO₄, and8.06 mM Na₂HPO₄ (D8662, Sigma-Aldrich, U.S.) and was allowed to re-foldon ice for 10 min. Prior to each round of selection, 2.5×10⁹ PFU mL⁻¹ ofVSV was incubated with 100 nM of FAM-labeled aptamer pool in a totalvolume of 50 μL (DPBS) for 30 min on a shaking incubator at 25° C. and400 r.p.m. The mixture was then centrifuged at 17 200 r.c.f. for 15 min.Next, the supernatant was discarded and 50 μL DPBS was added and themixture was centrifuged again. This washing step was repeated 3 timesfor rounds 1-5 and increased to 5 times for rounds 6-10. Upon completionof the last washing step, the pellet was re-suspended in 50 μL of anequimolar mixture of 2.5 mM EDTA (EMD Chemicals, U.S.)/EGTA (Bio BasicInc., Canada) in PBS (2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄ and137.93 mM NaCl) for 30 min. Afterward, the mixture was centrifuged for15 min at 17 200 r.c.f. and the supernatant was transferred to aseparate tube for storage at −20° C. Finally, aptamers were amplified byPCR and the cycle was repeated.

Aptamer pools were amplified using bundled symmetric and asymmetric PCRafter each subsequent round of selection. Symmetric PCR amplifies andproduces dsDNA, where 5 μL of the supernatant collected during selectionand containing the bound aptamers were mixed with 45 μL of symmetric PCRmaster mix. The master mix contained the following reagents in finalconcentrations: 1×PCR buffer (Promega Corporation, U.S.), 2.5 mM MgCl₂,0.028 U μL⁻¹ GoTaq Hot Start Polymerase (Promega Corporation, U.S.), 220μM dNTPs, 500 nM forward primer (5′CTCCTCTGACTGTAACCACG3′) (SEQ IDNO:1), and 500 nM reverse primer (5′GGCTTCTGGACTACCTATGC3′) (SEQ IDNO:20) (Integrated DNA Technology, U.S.). Upon completion, 5 μL of thesymmetric master mix were added to the asymmetric PCR master mixcontaining the same reagents as the symmetric master mix but with 1 μMforward FAM-labeled primer (FAM-5′CTCCTCTGACTGTAACCACG3′) (SEQ ID NO:1)and 50 nM reverse primer. Asymmetric PCR has low amplification power butit produces ssDNA. Both symmetric and asymmetric PCR used the followingprogram: preheating for 2 min at 95° C., 15 cycles for symmetric PCR or10-15 cycles for asymmetric PCR of 30 sec at 95° C., 15 s at 56.3° C.,15 s at 72° C., and hold at 4° C.

A total of to rounds of SwAps selection were performed, and the selectedaptamers were analyzed by flow cytometry.

Example 4: Flow Cytometric Affinity Analysis of Aptamer Pools and Clones

For affinity testing, pools were purified by loading the mixture onto 30kDa cut-off filter (Nanosep, U.S.). This was followed by centrifugationat 3 800 r.c.f. for 13 min at 16° C. Subsequently, an equal volume DPBSwas added for two additional washing steps for to min each. The puritywas tested by running the raw and purified samples on 3% agar gel(Sigma-Aldrich, U.S.) at 150V. Finally, concentration of sample wasmeasured using NanoDrop-2000 UV-Vis spectrophotometer, U.S.

Aptamer pool/clone affinity to VSV and switchability were measured usinga FC-500 Flow Cytometer (Beckman Coulter Inc., U.S.). All samples,contained 100 nM of purified FAM-labeled aptamer, were incubated with2.5×10⁷ PFU mL⁻¹ at room temperature for 30 min in DPBS. The sampleswere then divided into two portions; the first portion had DPBS added toit the second had to mM EDTA/EGTA 30 min at room temperature. Allsamples were made to 250 μL prior to flow analysis. Control experimentswere performed using the aptamer pool 8 and a sample of VSV was stainedusing TOTO-3 dye (Invitrogen, U.S.) to allow for identification on flowcytometry.

Ten selected pools of aptamers were examined for two criteria; theaffinity of the aptamer pool to VSV and the ability of this pool torelease VSV upon treatment with the EDTA/EGTA mixture which is denotedhere by the Coefficient of Switching (CoS). Rather than using the N40DNA library as a standard, it was decided to compare the aptamer poolsto the initial pool which was used to start selection. This was decidedas a better representation because unlike with typical SELEX protocolswhere the DNA library would represent “round 0”, here our starting poolwas pre-selected to bind to VSV. Thus, comparing to the native librarywould not have provided us with information as to whether the selectionscheme was truly creating switchable aptamers. All pools wereFAM-labeled, purified and made to a total volume of 100 μL in DPBS with50 nM aptamer pool and 10l PFU mL⁻¹ VSV. Flow cytometry results wereanalyzed by Kaluza software; one can see two trends in FIG. 2 referringto a weakly and strongly switchable aptamer pool. FIG. 2A shows thebinding of the to pools and control to VSV in the presence of Ca²⁺ andMg²⁺ (in blue) and amount of VSV remained bound to each pool afterincubation with EDTA/EGTA mixture (in red). Pools 3, 6, 9, and toexhibited strong affinity to VSV and are thus promising candidates.

-   -   a. FIG. 2B shows the CoS values calculated for each respective        pool, where a CoS of 1 indicates an aptamer exhibiting the        highest switchability, whereas 0 refers to an aptamer completely        unable to switch. Using equation 1:

$\begin{matrix}{{CoS} = {1 - \left( \frac{\%\mspace{14mu}{Bound}\mspace{14mu}{Aptamers}\mspace{14mu}{to}\mspace{14mu}{VSV}\mspace{14mu}{in}\mspace{14mu}{DPBS}}{{\%\mspace{14mu}{Bound}\mspace{14mu}{Aptamer}\mspace{14mu}{to}\mspace{14mu}{VSV}\mspace{14mu}{with}\mspace{14mu}{EDTA}} + {EGTA}} \right)}} & (1)\end{matrix}$

How effectively the aptamers can switch from their bound and unboundform can be compared. Round 0 is the lowest, followed by rounds 2, and8, all showing a CoS of <0.20. The CoS was small, which indicates thatthe binding of the pool to VSV was largely unaffected by the presence orabsence of Ca²⁺ and Mg²⁺. Large CoS values was exhibited by pools 3, 7and 10. Since rounds 1 and 2 represent the beginning of selection, itwas expected that they would not show a good switching functionality.One would think that as the number of rounds of selection increases, thebinding and switchability characters would also increase linearly. Thisis seldom the case in aptamer selection as after each round, mutationswere introduced during PCR, which may be beneficial or detrimental tobinding. Pool 10 showing the highest affinity and switchability was thusselected for cloning. More washing steps were employed during the laterrounds which resulted in more specificity. Flow cytometry histograms ofround to are demonstrated in FIG. 2C where the switchable nature of theaptamer pool is clear. When virus is bound by an aptamer, thefluorescence of the virus particle increases resulting in a shift to theright as seen in the histogram. Similarly, when the VSV-aptamer complexdissociates in the presence of EDTA/EGTA, the overall fluorescencedecreases and the histogram shifts to the left side.

Aptamer pool 10 was cloned and a total of 15 SwAps sequences wereobtained. All clones were tested for their respective affinities to VSVand the switchability as was described above. FIG. 3 shows the dataobtained for the clones using a Beckman FC600 flow cytometer. SwApsclones 1, 6, 11, and 15 exhibited good affinity and high CoS values. Inorder to confirm the data obtained using flow cytometry, anelectrochemical aptamer-based sensor was developed to estimate theaffinity between each aptamer and VSV as well as the switchability ofeach aptamer upon elution. This method had the added benefit of havingthe aptamers fixed on the surface of the gold sensor. This would likelymimic the behavior of aptamers when they would be covalently attached toan affinity chromatography matrix or streptavidin coated beads as inthis work, and allow for selection of aptamers that would show the bestperformance in the course of VSV purification.

Example 5: Cloning and Sequencing of High Affinity SwAps

Aptamer pool 10 (SEQ ID NO: 18) which showed both high affinity andswitchability was selected for cloning to obtain individual aptamersequences. The resulting sequences are provided in SEQ ID NOs: 3 through17, also reproduced below at Table 3.

Briefly, the pool was amplified using symmetric PCR to obtain dsDNA andthen was purified using a DNA gel extraction kit (AxyGen Biosciences,U.S.). Cloning was performed according to the protocol obtained with theM13mp18 perfectly blunt cloning kit (Novagen, U.S.). White coloniesidentifying the presence of the insert were then grown in LB+ Ampicillinovernight in a shaking incubator. PCR was then performed to ensure thepresence of the insert with the following master mix. For each PCRreaction, 2 μL of the cell suspension was mixed with 18 μL of the PCRMaster Mix containing: 1×PCR GC buffer, 2.5 mM MgCl₂ (MallinckrodtBaker, Inc., U.S.), 0.01 U μL⁻¹ KAPA 2G Robust Hot Start DNA polymerase(Kapa Biosystems, U.S.), 220 μM dNTPs, 0.5 μM forward FAM-labeledprimer, and 0.5 μM reverse primer. Amplification was performed using aPCR program (preheating for 5 min at 95° C., 30 cycles of 30 s each at95° C., 15 s at 56.3° C., 15 s at 72° C., and hold at 4° C.). Uponidentifying the clones with the plasmid containing the insert,amplification was performed. This was carried out using the sameaforementioned master mix but with the M13 forward primer(5′GTAAAACGACGGCCAGT3′) (SEQ ID NO:91) and M13 reverse primer(5′AGCGGATAACAATTTCACACAGG3′) (SEQ ID NO:92). The unpurified PCR productobtained was sent to McGill University and Génome Québec InnovationCentre, Canada for sequencing.

Example 6: Impedimetric Analysis of the Affinity and SwitchingProperties of SwAps

Electrochemical studies, including cyclic voltammetry (CV) andelectrochemical impedance spectroscopy (EIS) were carried out with anelectrochemical analyzer (CH Instruments 660D, TX, U.S.) connected to apersonal computer. All measurements were performed at room temperaturein an enclosed and grounded Faraday cage. A conventional three-electrodeconfiguration printed on a ceramic substrate; including a GNPs-SPCEelectrode as the working electrode, carbon counter electrode, and asilver pseudo-reference electrode. A three-electric contacts edgeconnector was used to connect the screen-printed electrode with thepotentiostat (Dropsens, Spain). The open-circuit or rest-potential ofthe system was measured prior to all electrochemical experiments toprevent sudden potential-related changes in the self-assembled monolayer(SAM). CV experiments were performed at a scan rate of 100 mV s⁻¹ in thepotential range from −600 to 800 mV. EIS measurements were conducted inthe frequency range of 100 kHz to 0.1 Hz, at a formal potential of 250mV and AC amplitude of 5 mV. The measured EIS spectra were analyzed withthe help of equivalent circuit using ZSimpWin 3.22 (Princeton AppliedResearch, U.S.) and the data were presented in Nyquist plots.Electrochemical measurements were performed in 25 mM sodium phosphatebuffer (pH 7), containing 2.5 mM K₄Fe(CN)₆ and 2.5 mM K₃Fe(CN)₆.

Prior to experiments, the gold nanoparticles modified screen-printedcarbon electrode (GNPs-SPCE) (L33×W10×H0.5, Dropsens, Spain) was washedthoroughly with deionized water then dried with pure N₂. Subsequently,the electrode was incubated with equimolar amounts (1 μM) of theselected aptamer and an HPLC purified,Thiol-spacer-5′GGCTTCTGGACTACCTATGC3′ (SEQ ID NO:20) modified at the 5′position with a 6-hydroxyhexyl disulfide group, detection probe(Integrated DNA Technologies, U.S.) in 25 mM sodium phosphate buffer, pH7, for 5 days at 4° C. Finally, the electrode was incubated with 1 mM2-mercaptoethanol in ethanol for 5 min to back-fill the empty spots ofthe electrode surface, thus reducing the non-specific adsorption ontothe surface.

The electrochemical characteristics of the developed impedimetric sensoris provided in FIG. 5 and Table 1.

TABLE 1 Equivalent circuit element values for the developed aptasensorafter each modification step Rs (Ω) CPE (μF) n R_(CT) (Ω) W (μF^(0.5))Bare electrode 74.17 4.95 0.85 5710 118 After formation of 73.7 2.560.91 6710 125 the thiolated hybrid After surface 70.18 3.04 0.92 7631170 backfilling with 2- mercaptoethanol

The rationale behind this electrochemical approach is that the bindingbetween the virus and the respective aptamer, will further block thecharge transfer from the solution-based redox probe to the electrodesurface. Consequently, R_(CT) will become increasingly high and can beused to monitor the binding event (Ayyar et al.; Wei et al.). Aschematic representation of the sensor preparation and performance isprovided in FIG. 4A. A hybrid of a thiol-modified primer and the SwApwas self-assembled onto a gold nanoparticles-modified screen-printedcarbon electrode. Binding of VSV to the immobilized SwAp, in thepresence of Ca²⁺ and Mg²⁺, causing an increase in the interfacialresistance from the baseline value R_(CTB) to R_(CTV). Incubation with amixture of EDTA and EGTA causes a switch in the aptamer conformationwith a subsequent release of VSV and a shift-back in the interfacialresistance to R_(CTS).

More specifically, incubation of 1×10⁶ PFU of VSV in Dulbecco'sphosphate buffered saline (DPBS) with the respective SwAp immobilizedonto the electrode surface for 1 hour at room temperature, caused anincrease in the interfacial resistance and consequently in the value ofR_(CT). Fifteen aptamer sequences were tested and the affinity betweenthe virus and each sequence was expressed as the change in R_(CT) value,which is calculated from the difference between R_(CT) after incubationwith the virus (R_(CTV)) and the baseline resistance obtained after thepreparation of the aptasensor, R_(CTS). Afterward, each aptasensor wasincubated with an equimolar mixture of EDTA and EGTA (50 mM) for 30 minat room temperature. As can be seen in FIG. 4B and FIG. 6, treatmentwith the EDTA/EGTA mixture caused a decrease in impedance. This could beascribed to the chelation of the Ca²⁺ and Mg²⁺ ions, which in turncaused a change in the conformation of the SwAp and consequently forcedthe virus to dissociate from its complex with the immobilized SwApleading to a parallel shift-back in impedance. FIG. 6 shows Nyquistplots (−Z_(im) vs. Z_(re)) of impedance spectra of VSV aptasensors basedon 15 aptamer clones (Swaps1→Swaps15) obtained (a) after aptasensorpreparation (b) after binding of 1×10⁶ PFU of VSV in Dulbecco'sphosphate buffered saline (DPBS), and (c) after treatment with anequimolar mixture of EDTA and EGTA (50 mM). A control experiment wasperformed, under the same conditions, using the original aptamer poolutilized in selection. The impedance spectra were recorded from 100 kHzto 0.1 Hz and the amplitude was 0.25 V vs. Ag pseudo-reference in 25 mMsodium phosphate buffer (pH 7), containing 2.5 mM K₄[Fe(CN)₆] and 2.5 mMK₃[Fe(CN)₆].

The switching ability of each SwAp can be expressed as the Coefficientof Switching (CoS), which can be calculated from the formula 2:

$\begin{matrix}{{CoS} = {1 - \left( \frac{{RCTS} - {RCTB}}{{RCTV} - {RCTB}} \right)}} & (2)\end{matrix}$

Where R_(CTS) represents the resistance to charge transfer after aptamerswitching due to EDTA/EGTA treatment. A control experiment wasperformed, under the same conditions, using the original aptamer poolemployed in selection. The circuit elements calculated for each SwAp areprovided in Table 2. In FIG. 4B, the control experiment was performedunder the same conditions, using the original aptamer pool utilized inselection.

TABLE 2 Equivalent circuit element values for the developed aptasensorsusing different SwAps, where SwAp_(B), SwAp_(V), and SwAp_(S) representsthe circuit elements before VSV binding, after incubation with 1 × 10⁶PFU of VSV, and after regeneration using EDTA/EGTA, respectively. R_(s)CPE R_(CT) W (Ω) (μF) n (Ω) (μF^(0.5)) SwAp_(B)1 74.02 2.26 0.92 6691185 SwAp_(V)1 72.6 1.8 0.94 7729 173 SwAp_(S)1 73.49 1.78 0.94 7581200.5 SwAp_(B)2 71.81 6.14 0.86 5731 202.6 SwAp_(V)2 70.58 3.43 0.917149 168.8 SwAp_(S)2 71.54 2.9 0.91 6794 181.4 SwAp_(B)3 75.45 3.32 0.97822 117.4 SwAp_(V)3 74.85 7.76 0.87 8625 7.067 SwAp_(S)3 72.27 3.34 0.98027 110.1 SwAp_(B)4 70.87 2.69 0.91 7016 80.35 SwAp_(V)4 71.78 2.680.91 7971 90.02 SwAp_(S)4 70.03 3.82 0.9 7427 88.04 SwAp_(B)5 73.6 4.720.91 6551 91.37 SwAp_(V)5 75.18 2.05 0.93 7389 191.5 SwAp_(S)5 72.592.14 0.93 6601 176.8 SwAp_(B)6 71.96 4.4 0.9 6625 113.1 SwAp_(V)6 72.362.22 0.93 7917 177.6 SwAp_(S)6 74.45 2.47 0.92 7497 213 SwAp_(B)7 74.933.42 0.9 7574 241.8 SwAp_(V)7 73.99 2.05 0.93 9346 319.6 SwAp_(S)7 73.212.34 0.93 8400 268.7 SwAp_(B)8 70.15 8.07 0.87 5454 184.3 SwAp_(V)868.52 3.74 0.9 7132 185.6 SwAp_(S)8 69.52 3.54 0.9 6128 188.1 SwAp_(B)971.12 3.12 0.91 6931 168.8 SwAp_(V)9 71.24 12.48 0.9 8111 87.31SwAp_(S)9 67.6 3.39 0.91 6952 156 SwAp_(B)10 73.83 8.77 0.87 4321 202.8SwAp_(V)10 73.21 3.46 0.9 8043 325.5 SwAp_(S)10 72.16 3.15 0.91 7923309.3 SwAp_(B)11 73.09 9.43 0.92 7269 81.4 SwAp_(V)11 73.47 2.31 0.937642 200 SwAp_(S)11 71.8 2.41 0.93 7338 185.5 SwAp_(B)12 70.73 4.88 0.95495 147.9 SwAp_(V)12 74.75 2.22 0.92 7670 272.9 SwAp_(S)12 72.96 2.110.93 7314 289.8 SwAp_(B)13 72.1 3.9 0.91 5269 243.2 SwAp_(V)13 73.612.19 0.93 7253 228.5 SwAp_(S)13 72.38 2.12 0.93 6905 200.5 SwAp_(B)1473.6 13.11 0.86 6590 111.7 SwAp_(V)14 71.41 2.29 0.93 7299 168SwAp_(S)14 72.33 2.48 0.92 6825 197.3 SwAp_(B)15 71.67 2.91 0.9 6940165.8 SwAp_(V)15 72.75 5.89 0.9 7870 95.6 SwAp_(S)15 70.65 2.89 0.917310 164.9 Pool_(B) 70.28 9.05 0.93 7248 95.31 Pool_(V) 72.35 12.12 0.927430 103.4 Pool_(S) 72.12 8.26 0.93 7327 129.3

As can be seen in Table 3, the values of R_(CTV)-R_(CTB) and CoS weredetermined for each aptamer sequence and both parameters were employedto assess the efficiency of each SwAp for VSV purification. In otherwords, SwAps exhibiting high virus affinity and switching ability,including SwAps clones 9, 5, 7 and 3, have the potential to be furtherintegrated into affinity chromatography units involved in VSVpurification. A slight variation of the results obtained using theimpedimetric sensor was observed when compared to the flow cytometrydata. This could be ascribed to the different forms of aptamers used ineach method where free aptamers were used in flow cytometry, whereasimmobilized aptamers were used to develop the sensors. Thus, they mayadopt different tertiary structures and alter their bindingcapabilities.

TABLE 3Sequences of switchable aptainers, their affinities to VSV expressed asthe change in charge transfer resistance after binding to the virus (R_(CTV)-R_(CTB)), and coefficient of switching (CoS) where F: 5′CTCCTCTGACTGTAACCACG3'(SEQ ID NO: 1) and R: 5′GCATAGGTAGTCCAGAAGCC3′ (SEQ ID NO: 2)Coefficient (R_(CTV)-R_(CTB)) of Switching Clone Sequence Ω (CoS) SwAp1F-CGC CCT CAG AAC TTT TGT ATC CGA ACA CCT 1038 0.14 SEQ IDGCA TCG TCC G-R NO: 3 SwAp2 F-TAC CAC CCG TGA CGC GCA CAT CCC TCC TCT1418 0.12 SEQ ID GTT CTC CGC G-R NO: 4 SwAp3F-TGC CCC CTC CAT CCC GAG TAA CCT ACG TCC  803 0.74 SEQ IDATG TCT CGC T-R NO: 5 SwAp4 F-TGC CCC CTC CAT CCC GAG TAA CCT ACG TCC 955 0.57 SEQ ID ATG TCT CGC T-R NO: 6 SwAp5F-TAC CAC CCG TGA CCC TCA CAT CCC TCC TCT  838 0.94 SEQ IDGTT CTC CGC G-R NO: 7 SwAp6 F-TGG CAC TGT TGT CAT CAC TGT CCC CCC CTA1292 0.33 SEQ ID ACT CGT CCG T-R NO: 8 SwAp7F-TAC CAC CCG TGG CCC TCA CAT CCC TCC TCT 1772 0.53 SEQ IDGTT CTC CGC G-R NO: 9 SwAp8 F-TAC CAC CCG TGA CCC TCA CAT CCC TCC TCT1678 0.6  SEQ ID GAC GTA ACC ACG CG-R NO: to SwAp9F-TAC CAC CCG TGGCCC TCA CAT CCC TCC TCT 1180 0.98 SEQ IDGTT CTC CGC G-R NO: 11 SwAp10 F-TAC CGC CCG TGA CCC TCA CAT CCC TCC TCT3722 0.03 SEQ ID GTT CTC CGC G-R NO: 12 SwAp11F-CAG CCA CCA TAC TGT CCC GTT TGC CCC CGC  373 0.82 SEQ IDCGA TTC CGT C-R NO: 13 SwAp12 F-TAC CAC CCG TGA CCC TTA CAT CCC TCC TCT2175 0.16 SEQ ID GTT CTC CGC G-R NO: 14 SwAp13F-TAC CAC CCG TGA CCC TCA CAT CCC TCC TCT 1984 0.18 SEQ IDGTT CTC CGC G-R NO: 15 SwAp14 F-TAC CAC CCT TGA CCC TCA CAT CCC TCC TCT 709 0.67 SEQ ID GTT CTC CGC G-R NO: 16 SwAp 15F-GCA CCC CGA CCC AAT TTC CCC CAT ACT TCA  930 0.6  SEQ IDTCC TGT TTC G-R NO: 17 Pool 10 F-NNN NNN NNN NNN NNN NNN NNN NNN  1820.57 SEQ ID NNN NNN NNN NNN NNN-R NO: 18

Example 7: Electrochemical Characterization of the Developed SwAps

The electrochemical characteristics of the developed aptasensor wereinvestigated by both CV and EIS. As shown in FIG. 5A, the pretreatedelectrode presents a quasi-reversible voltammogram indicating that theredox reactions easily occurred on the bare electrode surface, evidencedby the large redox currents (curve a). Formation of SAM of the thiolatedprimer (capture probe) hybridized with the VSV-specific SwAp (detectionprobe) on the electrode surface significantly reduced the electrodecurrent; also the sigmoidal behaviour observed in curve b could beindicative of limited charge transfer via tunneling or diffusion throughthe defects in the formed layer. Moreover, the repulsion between thenegatively charged DNA backbone and the redox probe, [Fe(CN)₆]^(3−/4−),is responsible of the significant reduction of the redox currents. Finaltreatment with 2-mercaptoethanol greatly reduced the redox currentsbecause they can penetrate down to the electrode surface, therebyblocking the direct access of the conducting ions (curve c). The Nyquistplots of the impedance spectra are shown in FIG. 5B and the circuitelement values are presented in Table 1, which support the CV data. Thecomplex impedance was presented as the sum of the real Z, Z_(re), andimaginary Z, Z_(im), components that originate mainly from theresistance and capacitance of the electrical cell, respectively. Asuitable equivalent circuit, shown in the inset of FIG. 5B, wascarefully selected to express the electrochemical process and to enablefit producing accurate values. A modified Randles circuit consists ofthe ohmic resistance; R_(S), of the electrolyte solution, the electroniccharge transfer resistance, R_(CT), in series with the finite lengthWarburg W, and in parallel with a constant phase element, CPE,associated with the double layer and reflects the interface between theassembled film and the electrolyte solution. The solution resistance,R_(S), is the resistance between the aptamer-modified electrode and thereference electrode. The high frequency semicircle of the Nyquistdiagram corresponds to the charge transfer resistance, R_(CT), inparallel with the CPE. The former represents the electron-transferkinetics of the redox probe at the electrode surface, whereas the lattercorresponds to a nonlinear capacitor accounting for the inhomogeneity ofthe formed film (Fitzgerald et al.). The diameter of the semicirclecorresponds to the interfacial resistance at the electrode surface, thevalue of which depends on the dielectric and insulating features of thesurface layer. On the other hand, the Warburg impedance, Z_(W), accountsfor a diffusion-limited electrochemical process, presumably due tomolecular motions within the film caused by conducting ions penetration(Yang et al.).

Example 8: Effects of Buffer on Binding

The switching ability of aptamer pools was hypothesized to stem from theremoval of Ca²⁺ and Mg²⁺ which can bind to phosphate backbone of ssDNA.To confirm that this was indeed the case and not the result of a changein buffer ionic strength, a flow cytometric analysis was performed asshown in FIG. 7. More specifically, FIG. 7 is a bar chart of the bindingaffinity of SwAp6 clone in different buffers. Aptamers (50 nM) incubatedwith VSV (10⁷ PFU) for 30 min prior to separation into 3 fractions. Each50 μL fraction was mixed with 250 μL of DPBS (MgCl₂ and CaCl₂), PBS orPBS with 10 mM EDTA/EGTA. SwAps clone 6 was allowed to bind to VSV for30 minutes and then separated and incubated in 3 different buffers.Results were obtained using flow cytometry and measuring fluorescence ofthe virus particles. It was observed that the amount of VSV bound to theSwAps has increased significantly in the presence of divalent cations.However, in instances when divalent cations were not present in thebuffer such as in PBS, the amount of virus bound to aptamer decreases.Finally, in PBS containing 10 mM of EDTA/EGTA, the amount of bound VSVwas the lowest due to the chelation of the divalent cations. This trendhas been noted before by others, where the effects of ionic strength andpH were examined with respect to aptamer-protein binding (Jiang et al.).

Example 9: SwAps-Based Purification of VSV

Briefly, FIG. 1B shows a schematic representation of virus purificationby SwAps in accordance with the method of the present disclosure.

In a low DNA binding tube, 200 nM of 5, 6, 7 and 9 clones were mixedtogether with either biotin-labeled forward(5′CGTGGTTACAGTCAGAGGAG3′/3Biotin) (SEQ ID NO: 19) or reverse(5Biosg/5′GGCTTCTGGACTACCTATGC3′) (SEQ ID NO: 20) complementary primers.An annealing protocol was used to hybridize the probes as follows; heatto 95° C. for 2 min and then gradually decrease the temperature to 20°C. by 1° C. every 3 sec. Streptavidin-coated magnetic beads (Cat#Z5482,Promega, U.S.) were used for the purification procedure. Briefly, 1 mLof beads was added to low DNA binding tube and the buffer was exchangedwith penicillin-streptomycin (P4333, Sigma-Aldrich, U.S.) and incubatedfor 2 hrs at 37° C. The beads were then washed with sterile DPBS,followed by the addition of 200 nM aptamer clone mixture and incubationat 25° C. for 2 hrs (shaking every 10 min to resuspend beads). Amagnetic stand (Z5331, Promega, U.S.) was used to separate the beadsfrom the aptamer mixture and the beads were then washed 3 times withDPBS. A mixture of VSV and cell debris collected from the VSV harvestingprocedure was mixed with aptamer-bead complex for 1 hr. Subsequently,the beads were washed 3 times with DPBS followed by the addition of 25mM EDTA/EGTA and incubation for 30 min at 25° C. This allowed for theliberation of VSV which was examined using flow cytometry and viralplaque assays.

For the selection of SwAps with switchable affinity to VSV, the mixtureof SwAps clones contained equal amount of 5, 6, 7, and 9 with eitherforward or reverse biotin primers. These clones were chosen because 5, 7and 9 showed promising results when assessed using the developedaptasensors. The conjugation of SwAps to magnetic beads mimics theapproach used for aptasensor analysis and thereby would suggest that theSwAps selected via electrochemical impedance spectroscopy would functionwell for purification. Although SwAps6 showed modest results in theaptasensor analysis, it was selected to be included among the best SwApsdue to its superior performance in free form as indicated by the flowcytometry data.

Biotinylated aptamers (SwAps6) are conjugated to streptavidin coatedmagnetic beads and mixed with VSV and cell debris solution obtained fromthe virus harvesting protocol. SwAps bind to VSV. Debris is removed bywashing the beads with DPBS. EDTA and EGTA are added to chelate themagnesium and calcium ions causing a conformational change whichreleases VSV. VSV is then collected.

As shown in FIG. 8, flow cytometry was used to assess the ability ofSwAps-coated magnetic beads to purify VSV from its mixture with celldebris. FIG. 8A shows the harvested VSV mixture which contained both thecell debris and VSV before purification. Upon incubation of the virusmixture with streptavidin-coated magnetic beads conjugated with amixture of SwAps, one can see in FIG. 8B that pure VSV was obtained fromthe mixture. Finally, to ensure that the gate titled VSV did contain thevirus, anti-VSV antibodies labeled with Alexa 547 was added and thefluorescence histogram of FIG. 8C clearly shows that VSV was collected.The SwAps clones have been selected so that they have an increasedsensitivity to the loss of divalent cations, the switching property hasbeen achieved with using 25 mM EDTA/EGTA. Thus, one limitation of thismethod is that the VSV collected is in a solution of EDTA/EGTA and mayrequire an exchange of buffer for further use. Despite this limitation,the method has proven successful as a potential procedure to purify VSVfrom contaminating material and is re-usable as the aptamer can reformthe tertiary structure when snap cooled (denatured and then cooledquickly to 4° C.) in DPBS.

The analysis was carried out using a plaque forming assay as seen inFIG. 9 where a 33% yield was obtained from the purification scheme.Specifically, FIG. 9 shows the plaque forming assay with infection ofVero cells with VSV, after VSV has undergone SwAps-based purification.Panel 1 shows DPBS alone, panel 2 shows infection of 100 PFU from stockVSV and panel 3 shows VSV retrieved upon completion of the purification.There is no infection seen in panel 1 containing DPBS alone. Panel 2shows 5.3×10⁶ PFU mL-VSV and panel 3 has 1.8×10⁶ PFU ml-liberated VSV.The resulting VSV recovered from the cell debris was 33.96%. 100%recovery would indicate the total activity of pure VSV before mixing itwith cell debris and purification with the method described herein.

Example 10: Cloning and Sequencing of High Affinity SwAps to Neuropilin1 (NRP) Receptor

An aptamer which showed both high affinity and switchability wasselected for cloning to obtain individual aptamer sequences to the NRPreceptor Pool. This pool was designed to be specific to NRP positivecells. The resulting sequences are provided in SEQ ID NOs: 21 through30, also reproduced below at Table 4. Briefly, the pool was amplified,purified and cloned using the same method as described in Example 5above.

TABLE 4 NRP- CTCCTCTGACTGTAACCACGGCGTGCCTATGGTGCGTGTGCCAAGTGT SMG-20GCGTGTAATGACATTCGTGAAAAGTGCGCGGGCATAGGTAGTCCAGAAG SEQ ID CC NO: 21 NRP-CTCCTCTGACTGTAACCACGGCGCGCGTTTGCACATGTGCGTGCGACAT SMG-19ATGCGTGGGGGGAGATGTATGAGACGTGTGGGCATAGGTAGTCCAGAA SEQ ID GCC NO: 22 NRP-CTCCTCTGACTGTAACCACGGCGCGTTCCTGGTAGCTCATGCGTGGCGT SMG-4GGACACATGCAGGTGCGGGTIGCCTGTGTGGGCATAGGTAGTCCAGAA SEQ ID GCC NO: 23 NRP-CTCCTCTGACTGTAACCACGCGAGTCGCCTACGTACGCACACTTACCGCG SMG-12CACGTCCGGGCAGGCGTGTCCCGTGCATGCGCATAGGTAGTCCAGAAGC SEQ ID C NO: 24 NRP-CTCCTCTGACTGTAACCACGGTGTACACTGGCACACGCACATGTCATCCG SMG-6CGCGGGGCTGCACACGTCAGCCGTGTGTGGGCATAGGTAGTCCAGAAG SEQ ID CC NO: 25 NRP-CTCCTCTGACTGTAACCACGGCGTACGTGACCCCATACGCACTCTAGCCA SMG-14CATACGTTCGCCCACGCCTGTCGCTCGCGGGCATAGGTAGTCCAGAAGC SEQ ID C NO: 26 NRP-CTCCTCTGACTGTAACCACGGTGTGCCTGCTTGCTGATGTGTGTTGTCG SMG-1GTGCGTGAGGGCGTACGTAAGTGTCGTGCGGGCATAGGTAGTCCAGAA SEQ ID GCC NO: 27 NRP-CTCCTCTGACTGTAACCACGGTCTGCACTATGGTGCGTGCGTTGGTGT SMG-E1ACCCATGAGCGTCCATGTGTGAGTTGCTCGGGCATAGGTAGTCCAGAAG SEQ ID CC NO: 28 NRP-CTCCTCTGACTGTAACCACGGTCTGCACTATGGTGCGTGCGTTCGGTGT SMG-E2ACCCATGAGCGTCCATGTGTGAGTTGCTCGGGCATAGGTAGTCGAGAAG SEQ ID CC NO: 29 NRP-CTCCTCTGACTGTAACCACGCTACATGTGAGGGCGCTTGCATGCAATAT SMG-E3GCAGACTCTGACGCGTGTGTTGGTTGTGTGGGCATAGGTAGTCCAGAAG SEQ ID CC NO: 30

Flow cytometry data indicated the aptamers that demonstrated the bestswitching capability. These are NRP-SMG-E1; NRP-SMG-E2 and NRP-SMG-E3.This data is reproduced at FIGS. 10A-F

Example 11: Cloning and Sequencing of High Affinity SwAps to LeukemiaInhibitory Factor (LIF) Receptor

An aptamer pool selected specifically to LIF positive cells and whichshowed both high affinity and switchability was selected for cloning toobtain individual aptamer sequences to the NRP receptor. The resultingsequences are provided in SEQ ID NOs: 31 through 47, also reproducedbelow at Table 5. Briefly, the pool was amplified, purified and clonedusing the same method as described in Example 5 above.

TABLE 5 LIF- CTCCTCTGACTGTAACCACGGTAGCTATGGCCACGTGCACATTCAGTATG SMG-1CACGTTAATGCTCGCATGTCGTACGCGTGGGCATAGGTAGTCCAGAAGCC SEQ ID NO: 31 LIF-CTCCTCTGACTGTAACCACGCCACCCGTCTTTGTGCATGCTTGTACTGCAT SMG-12ACATCTCGCCACACGCGTACAGCACACGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 32 LIF-CTCCTCTGACTGTAACCACGGGCATAGGCGGGTGTGTATCTGCCAAGCGC SMG-5GTGCTTGCTGATTCTCGCGCGAATCACAGGCGCATAGGTAGTCCAGAAGC SEQ ID C NO: 33 LIF-CTCCTCTGACTGTAACCACGGTGCAGGTGAGAGCATGTGCGTGTCATGGT SMG-8CGAMCGTGGCGCTTGCATTGGGTGTGCGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 34 LIF-CTCCTCTGACTGTAACCACGGCGTACATCCCCACACGTGCGTATTACGTG SMG-9CTCCCCCGTGCGTGTCGGTGGAGCGTGTGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 35 LIF-CTCCTCTGACTGTAACCACGCTGCATCCTAGGGTCTATGCCTAGGGGGCT SMG-10GCTATGCGTGCACGCGTGTCGGTCATGTGGGCATAGGTAGTCCAGAAGC SEQ ID C NO: 36 LIF-CTCCTCTGACTGTAACCACGGCATGTTTCCCCGCGTGTGCATTTGACGTG SMG-21TGTGTCCCCACGCACGTATCACGCAAGGGGGCATAGGTAGTCCAGAAGC SEQ ID C NO: 37 LIF-CTCCTCTGACTGTAACCACGGCGTGCACCTCCGCGTATGGCTTGCATATG SMG-11AGTGCTGTTCTCCGTATTTCGGACATACGGGCATAGGTAGTCCAGAAGCC SEQ ID NO: 38 LIF-CTCCTCTGACTGTAACCACGGCACATATCTTGCTGCCCACGTGCCACCACC SMG-16GTGTCTCCCTGCCCATCCGAAGTGCGCGCGCATAGGTAGTCCAGAAGCC SEQ ID NO: 39 LIF-CTCCTCTGACTGTAACCACGGCACCTGAGTCTGTCCGTCCGCTTGACACG SMG-CACGCAAGGGTATGCGCATCCCACACGCGCGCATAGGTAGTCCAGAAGC E46 SEQ C ID NO: 40LIF- CTCCTCTGACTGTAACCACGGCGCGTATCCCCGAGTGCGTACGCGGTGTT SMG-E8TGCTCGATCGTACGTGCATGGTGTGCGTGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 41 LIF-CTCCTCTGACTGTAACCACGGCGTGTGTCCCGGTGTGCGCATAGTCCAAG SMG-E16TACGTCGCCGTGTGTACGTTCAATGCGTGGGCATAGGTAGTCCAGAAGC SEQ IDCTCCTCTGACTGTAACCACGGTGCGTATGGACACGTCTGTACTGAGTGCG NO: 42CATGYYGAGACGCATGCGTCGTGCGTGTGTGCATAGGTAGTCCAGAAGC C LIF-CTCCTCTGACTGTAACCACGGTGCGTATGGACACGTCTGTACTGAGTGCG SMG-E6CATGTTGAGACGCATGCGTCGTGCGTGTGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 43 LIF-CTCCTCTGACTGTAACCACGGTGCATGTGTCGGTATGCGGGCCGCTTGTG SMG-E45CGTGTGACGACTCGTGTGTGTAATGCGCGCGCATAGGTAGTCCAGAAGC SEQ ID C NO: 44 LIF-TCCTCTGACTGTAACCACGCCATGCACCCGTGTGTGTGTGGTGTACGTGT SMG-E13GTGTCCACGGGAACGTATCACGTCATAGGGCATAGGTAGTCCAGAAGCC SEQ ID: 45 LIF-CTCCTCTGACTGTAACCACGGCGCGTGCGTAGGCATAGGTGTCGTGTACG SMG-E7CGTGTCTCAGCGCAATTGCGTCGGGTGTGTTCATAGGTAGTCCAGAAGC SEQ ID C NO: 46 LIF-CTCCTCTGACTGTAACCACGGCATGCGGTCTCGCACTCGGTTCTAGTGTC SMG-E5CACGCTTGTGTATGCGTGCGCGGTGTGTGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 47

Flow cytometry data showed that the aptamers with the best switchingcapability are LIF-SMG-E46, E8, E16, E6, E45, E13, E7 and E55. This datais reproduced at FIGS. 11A-J.\

Example 12: Cloning and Sequencing of High Affinity SwAps to Patched 1(PTCH1) Receptor

An aptamer pool selected specifically to PTCH1 positive cells and whichshowed both high affinity and switchability was selected for cloning toobtain individual aptamer sequences to the NRP receptor. The resultingsequences are provided in SEQ ID NOs: 48 through 59, also reproducedbelow at Table 6. Briefly, the pool was amplified, purified and clonedusing the same method as described in Example 5 above.

TABLE 6 PTCH₁- CTCCTCTGACTGTAACCACGCCGCGAGTTGCCACACATGCACTTCTCACACSMG-17 ATACCCGTGTACACGTACAGCACATATGCGCATAGGTAGTCCAGAAGCC SEQ ID NO: 48PTCH₁- CTCCTCTGACTGTAACCACGCCGCGAGTTGCCACACATGCACTTCTCACAC SMG-22ATACCCGTGTACACGTACAGCACATATGCGCATAGGTAGTCCAGAAGCC SEQ ID NO: 49 PTCH₁-CTCCTCTGACTGTAACCACGCCCAATCCCGCACCACGTGCATGCCACGCCC SMG-16ACGCATGAGTACACACGTACGGCGCATGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 50 PTCH₁-CTCCTCTGACTGTAACCACGCCCAATCCCGCACCACGTGCATGCCACGCCC SMG-21ACGCATGAGTACACACGTACGGCGCATGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 51 PTCH₁-CTCCTCTGACTGTAACCACGGTGCCTGCAGGGACGCGTGTAACCGGAATG SMG-4TACGCCGCGACGCACACGCCTAGTGTACGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 52 PTCH₁-CTCCTCTGACTGTAACCACGGTGCCTGCAGGGACGCGTGTAACCGGAATG SMG-11TACGCCGCGACGCACACGCCTAGTGTACGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 53 PTCH₁-CTCCTCTGACTGTAACCACGGTACGGTCGTCACTGTGCGTACGCTGTGCA SMG-18AAGATGCAAGTGCGCATACTGGGTGTCGGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 54 PTCH₁-CTCCTCTGACTGTAACCACGGTACGGTCGTCACTGTGCGTACGCTGTGCA SMG-23AAGATGCAAGTGCGCATACTGGGTGTCGGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 55 PTCH₁-CTCCTCTGACTGTAACCACGGGACACGCCGGGACGTGCATACCGGATGCG SMG-24CACGTAATCACCTGTGGGTGGGACGAGCCGGCATAGGTAGTCCAGAAGCC SEQ ID NO: 56 PTCH₁-CTCCTCTGACTGTAACCACGACGCGCGATGCGGCAAGCATGTTACGCCCA SMG-E1TGTATCTTCGTGCACATGCCCTCCGTGTGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 57 PTCH₁-CTCCTCTGACTGTAACCACGACACTGTCCTCCTCTGACTGTAACCACGGCA SMG-E2TAGGTAGTCCAGAAGCC SEQ ID NO: 58 PTCH₁-CTCCTCTGACTGTAACCACGACGCGCGATGCGGCAAGCATGTTACGCCCA SMG-E3TGTATCTTCGTGCACATGCCCTCCGTGTGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 59

Flow cytometry data shows that aptamers PTCH1-SMG-E1, E2 and E3 have thebest switching capability. This data is reproduced at FIGS. 12A-G

Example 12: Cloning and Sequencing of High Affinity SwAps to Delta-LikeLigand 4 (DLL4) Receptor

An aptamer pool selected specifically to DLL4 positive cells and whichshowed both high affinity and switchability was selected for cloning toobtain individual aptamer sequences to the NRP receptor. The resultingsequences are provided in SEQ ID NOs 60 through 74, also reproducedbelow at Table 7. Briefly, the pool was amplified, purified and clonedusing the same method as described in Example 5 above.

TABLE 7 DLL₄- CTCCTCTGACTGTAACCACGGCGCGTGCGGTTGAACATGTCCCC SMG-10TGTACCCGTGCCCGATCGTGTGTGTGGGGTGTGCGGGCATAGGT SEQ ID AGTCCAGAAGCC NO: 60DLL₄- CTCCTCTGACTGTAACCACGGTGCGCGTGCGAGTCTGCGCGTCC SMG-21TGCACATGTGTGTGTGTGTGTGCGTTCGGCGTGCGGGCATAGGT SEQ ID AGTCCAGAAGCC NO: 61DLL₄- CTCCTCTGACTGTAACCACGTCGGGTGATGCGGCGCACACACCG SMG-3TGGCCACGTGCCAAGGTGTGTCTTTGCTGTGCGTGCGCATAGGT SEQ ID AGTCCAGAAGCC NO: 62DLL₄- CTCCTCTGACTGTAACCACGGCATGAGTTGGGGTACCAATGTGT SMG-25ATTACGTATGCGTCGGGACACGAGTCTAATGTGTGTGCATAGGT SEQ ID AGTCCAGAAGCC NO: 63DLL₄- CTCCTCTGACTGTAACCACGGTGTGCGCGTTGCTACATGTTCGTT SMG-24CTGCGGGCGGTGAGGTTCGTATGTTGTGTCCGTGTGCATAGGTA SEQ ID GTCCAGAAGCC NO: 64DLL₄- CTCCTCTGACTGTAACCACGGCGCGTGTGGAGGCGTACACGTAG SMG-19CGCATCAGTGTCAGAGCATGTATACGGTGCATGTGAGCATAGGT SEQ ID AGTCCAGAAGCC NO: 65DLL₄- CTCCTCTGACTGTAACCACGACGGGTTTCGCCGCGTACATATCGA SMG-E25GTGGATGTGCTGCCGGGCGCTCTTCTCGTGCTCGTGCATAGGTA SEQ ID GTCCAGAAGCC NO: 66DLL₄- CTCCTCTGACTGTAACCACGATGCGTGTTGTCATGCGCGTACAG SMG-GGTGCACGTGTACTCATGCGTGTGTATATCGTGTGTGCATAGGT E43 SEQ AGTCCAGAAGCC ID NO:67 DLL₄- CTCCTCTGACTGTAACCACGGCCCGTGCGCCAATACAACTGTGCA SMG-ATGTGTGTGCCGCTGTGTCTTCTTCCGGCGTGTGTGCATAGGIA E69 SEQ GTCCAGAAGCC ID NO:68 DLL₄- CTCCTCTGACTGTAACCACGTCGTGTGTGTGGGTGTACGCATTCT SMG-GTGCGCGTACCAGGCCACGCACGTCTCGCCTGTGTGCATAGGTA E24 SEQ GTCCAGAAGCC ID NO:69 DLL₄- CTCCTCTGACTGTAACCACGGTACACATAGCCATGTGAGCGCGC SMG-E76CGCGTGGATGTCCGCACTCATGCGTTTCGTACGTGCGCATAGGT SEQ ID AGTCCAGAAGCC NO: 70DLL₄- CTCCTCTGACTGTAACCACGCCATGAACCGTGGCCCCTGCATCGC SMG-E31GCATATGTGTGATAGTGTGTGTGCTCTCCGCCTGGGCATAGGTA SEQ ID GTCCAGAAGCC NO: 71DLL₄- CTCCTCTGACTGTAACCACGGCGCGCGCACCAATGTACGCATATT SMG-E9TTGCTCGTATAGGTTTCCCTGCGTTGACTGTGTGGGCATAGGTA SEQ ID GTCCAGAAGCC NO: 72DLL₄- CTCCTCTGACTGTAACCACGACGGGTACGTAGATCCGCGTATCG SMG-E7CGTGTAGGTACCGGGGTTCGTTGATCGAGTGTGTGCGCATAGGT SEQ ID AGTCCAGAAGCC NO: 73DLL₄- CTCCTCTGACTGTAACCACGGCACGCATATCAGTGCACACATCGC SMG-E1ACACATGCACGCGAAAACCTGGGCCGCATGTGTGGGCATAGGTA SEQ ID GTCCAGAAGCC NO: 74

Flow cytometry data showed that DLL4-SMG-E25, E43, E69, E24, E76, E31,E9, E7 and E1 had the best switching capability. This data is reproducedat FIGS. 13A-F

Example 14: Cloning and Sequencing of High Affinity SwAps to PlasminogenActivator, Urokinase Receptor (PLAUR)

An aptamer pool selected specifically to PLUR/PLAUR) positive cells andwhich showed both high affinity and switchability was selected forcloning to obtain individual aptamer sequences to the NRP receptor. Theresulting sequences are provided in SEQ ID NOs: 75 through 89, alsoreproduced below at Table 8. Briefly, the pool was amplified, purifiedand cloned using the same method as described in Example 5 above.

TABLE 8 PLUR- CTCCTCTGACTGTAACCACGCATAGGTAGTCCAGAAGCCAGCCTCCTTTG SMG-72ACTGTAACCACGGCATAGGTAGTTCAGATGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 75 PLUR-CTCCTCTGACTGTAACCACGGCATGTGTACCGGTGTATGCATGCAGCGCA SMG-95CATGTTCCCGAATGTGCGTCGAGTGCGCGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 76 PLUR-CTCCTCTGACTGTAACCACGGCATGTTCGGTAGCGCGTATGTGCAGTTCG SMG-62CGTGTTTATGCCTCGACGTAGTGTGCGCGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 77 PLUR-CTCCTCTGACTGTAACCACGCCATACTTGGTGGTCTGTGCGTAGAGGCGAG SMG-5TGTGCATCGGCATGCGTCTGCGCTGTGCGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 78 PLUR-CTCCTCTGACTGTAACCACGACGTGTGCCCGGGTGAACCGGCGCAGCGC SMG-29GTGTATGGTTATGCATGTGTCAGGTCCGTGCGCATAGGTAGTCCAGAAG SEQ ID CC NO: 79 PLUR-CTCCTCTGACTGTAACCACGACGCACTTTTGGGGTTGGTATGCGGGGTGCG SMG-66CACACGTCCGGACATGTGTCCTTCGTTCGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 80 PLUR-CTCCTCTGACTGTAACCACGGCATGCGTCAGCATGGGTGCATCCAGCGTG SMG-113CGCGTCGAAGGATGTGAATCTTGTGTATGCGCATAGGTAGTCCAGAAGC SEQ ID C NO: 81 PLUR-CTCCTCTGACTGTAACCACGACACATGCAGTGGTGTTTGTGTCATGCGTA SMG-25CATGTCTACGTGTGCGAGTTTGATGCGCGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 82 PLUR-CTCCTCTGACTGTAACCACGATGCGCGTTCGTGTGCGTAGGTFGGGTATG SMG-27TGCGTTTGAGTATGTGGACGTCGTGTGGGGGCATAGGTAGTCCAGAAGC SEQ ID C NO: 83 PLUR-CTCCTCTGACTGTAACCACGCTCTGTGGCGTTATGCGCGTGTCCAGTGTG SMG-TTCCCTGACATGTATGAGTTCGATACGCGGGCATAGGTAGTCCAGAAGCC E50 SEQ ID NO: 84PLUR- CTCCTCTGACTGTAACCACGGCGTCGGAGTGTGCATGTTCGTCTGATGCG SMG-E13CGGATGTCTCCTCATGTGTCGTGCGTATGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 85 PLUR-CTCCTCTGACTGTAACCACGGCACACGATTAGGCGCGGGGACCCTGTGTG SMG-E76TATCGCGTGATACGTATGCGCAGTACGCGTGCATAGGTAGTCCAGAAGCC SEQ ID NO: 86 PLUR-CTCCTCTGACTGTAACCACGGTGTATGTGGCTGTAGGTGCGTGCGGTTTG SMG-E35TGTGTCACGGTAAGCTTGCCCGGTGTGTGTGCATAGGTAGTCCAGAAGC SEQ ID C NO: 87 PLUR-CTCCTCTGACTGTAACCACGATACGGGTAAACGCGAGCGTGCATGAAGTG SMG-ATTGACGGCGCAGGCCTGTGGAGTGGGCAGGCATAGGTAGTCCAGAAGC E20 SEQ C ID NO: 88PLUR- CTCCTCTGACTGTAACCACGGAGTGCGTGGCTAAGCGCGTCTCGGGTTTC SMG-E31CATATTGCTGTGTGTGCATCCACCATGTGCGCATAGGTAGTCAGAAGCC SEQ ID NO: 89

Flow cytometry data indicated that PLAUR-SMG-E50, E13, E76, E35, E20 andE31 had the best switching capability. This data is reproduced at FIGS.14A-I. PLAUR titration data is reproduced at FIGS. 15A-H.

The scope of the disclosure should not be limited by the embodiments setforth in the examples but should be given the broadest interpretationconsistent with the description as a whole. The claims are not to belimited to the preferred or exemplified embodiments of the disclosure.

REFERENCES

To the extent that external references may be incorporated by referenceinto the present specification, all references identified herein areincorporated into this specification by reference in their entirety.

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The invention claimed is:
 1. A method of isolating or selecting aswitchable aptamer having affinity for a target ligand from a poolcomprising a mixture of aptamers, the method comprising the steps of: a)incubating said pool with said target ligand and a binding ion to formtarget-aptamer complexes comprising said target ligand and aptamersspecific to said target; b) separating unbound aptamer molecules fromthe target-aptamer complexes; c) contacting the target-aptamer complexeswith a chelating agent having affinity for said binding ion wherein aswitchable aptamer specific to said target is released from thetarget-aptamer complexes; and d) isolating or selecting the switchableaptamer released in step c.
 2. The method of claim 1 wherein at leastsaid steps a through c are performed at a maximum temperature of 25° C.3. The method of claim 1 comprising the further step of amplifying saidswitchable aptamer isolated in said step d.
 4. The method of claim 1further comprising the step of measuring the affinity of said switchableaptamer for the target in the presence and absence of the binding ion.5. The method of claim 1 wherein two or more switchable aptamers areisolated or selected and said steps a through d are repeated using thetwo or more switchable aptamers in place of the mixture of aptamers insaid pool wherein a switchable aptamer is isolated or selected which hasan increased affinity for the target ligand relative to others of saidswitchable aptamers.
 6. The method of claim 1 wherein the target ligandis a virus, a cell or an antibody.
 7. The method of claim 6 wherein thevirus is Vesicular Stomatis Virus (VSV).
 8. The method of claim 6wherein said cell is receptor-positive for one or more of the followingreceptors: a Neuropilin 1 (NRP) receptor, a Leukemia inhibitory factor(LIF) receptor, a Patched 1 (PTCH1) receptor, a Delta-Like Ligand 4(DLL4) receptor or a plasminogen activator/urokinase receptor (PLAUR).9. The method of claim 1 wherein the binding ion is calcium or magnesiumor a combination thereof.
 10. The method of claim 1 wherein thechelating agent is ethylene diamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA) or a combination thereof.
 11. The methodof claim 1 wherein the target-aptamer complexes and the unbound aptamermolecules are separated by centrifugation or by immobilizing thetarget-aptamer complexes and washing away unbound aptamer.
 12. Themethod of claim 1 wherein said pool comprises a randomized pool ofaptamers.
 13. The method of claim 1 wherein said aptamers in said poolcomprise oligonucleotides having a randomized region.
 14. The method ofclaim 13 wherein said randomized region is from about 20 and about 60nucleotides.
 15. The method of claim 14 wherein said randomized regionis about 40 nucleotides.
 16. The method of claim 8 wherein the targetligand is a cell that is receptor-positive for NRP receptor, and theaptamer pool comprises one or more of aptamers NRP-SMG-E1 (SEQ ID NO:28), NRP-SMG-E2 (SEQ ID NO: 29) and NRP-SMG-E3 (SEQ ID NO: 30).
 17. Themethod of claim 8 wherein the target ligand is a cell that isreceptor-positive for LIF receptor, and the aptamer pool comprises oneor more of aptamers LIF-SMG-E46 (SEQ ID NO: 40), LIF-SMG-E8 (SEQ ID NO:41), LIF-SMG-E16 (SEQ ID NO: 42), LIF-SMG-E6 (SEQ ID NO: 43),LIF-SMG-E45 (SEQ ID NO: 44), LIF-SMG-E13 (SEQ ID NO: 45), LIF-SMG-E7(SEQ ID NO: 46) and LIF-SMG-E55 (SEQ ID NO: 47).
 18. The method of claim8 wherein the target ligand is a cell that is receptor-positive forPITCH1 receptor, and the aptamer pool comprises one or more of aptamersPTCH1-SMG-E1 (SEQ ID NO: 57), PTCH1-SMG-E2 (SEQ ID NO: 58) andPTCH1-SMG-E3 (SEQ ID NO: 59).
 19. The method of claim 8 wherein thetarget ligand is a cell that is receptor-positive for DLL4 receptor, andthe aptamer pool comprises one or more of aptamers DLL4-SMG-E25 (SEQ IDNO: 66), DLL4-SMG-E43 (SEQ ID NO: 67), DLL4-SMG-E69 (SEQ ID NO: 68),DLL4-SMG-E24 (SEQ ID NO: 69), DLL4-SMG-E76 (SEQ ID NO: 70), DLL4-SMG-E31(SEQ ID NO: 71), DLL4-SMG-E9 (SEQ ID NO: 72), DLL4-SMG-E7 (SEQ ID NO:73) AND DLL4-SMG-E1 (SEQ ID NO: 74).
 20. The method of claim 8 whereinthe target ligand is a cell that is receptor-positive for PLAURreceptor, and the aptamer pool comprises one or more of aptamersPLAUR-SMG-e50 (SEQ ID NO: 84), PLAUR-SMG-E13 (SEQ ID NO: 85),PLAUR-SMG-E76 (SEQ ID NO: 86), PLAUR-SMG-E35 (SEQ ID NO: 87),PLAUR-SMG-E20 (SEQ ID NO: 88) and PLAUR-SMG-31 (SEQ ID NO: 89).
 21. Themethod of claim 1 wherein said pool comprises the nucleotide sequence ofSEQ ID NO:
 18. 22. The method of claim 16 wherein the aptamer sequencesin the pool comprise SEQ ID NOs: 3 though 17.